Cytokines regulate cellular growth and differentiation, hematopoiesis metabolism, and tissue repair. These factors are also critical in host defense, but are also major contributors to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, lupus, IBD, psoriasis, as well as allergy and asthma, underscoring the need to better understand the molecular basis of cytokine action and their regulation of expression. This year we made efforts to better understand the control of a cytokine that is of interferon-gamma (IFN-g), the canonical mediator of type 1 responses. Downstream of the Ifng gene is a long noncoding RNA (lncRNA) designated Ifng-as1. To study this locus, we generated two genetic models (1). DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. These results establish Ifng-as1 as an important regulator of Ifng expression, arguing for the need for further investigation of this interesting lncRNA. Type 2 responses encompass production of cytokines including interleukin (IL)-4, IL-5 and IL-13, which are important for elimination of helminths as well as the immunopathology of allergy and asthma. Innate lymphocytes maintain tissue homeostasis at mucosal barriers, with group 2 innate lymphoid cells (ILC2s) producing type 2 cytokines to control helminth infection. We used single-cell analysis to explore the diversity of gene expression among lung lymphocytes during helminth infection and identified a subset of ILC2s that preferentially expressed IL-5, together with the neuropeptide Calca-encoding calcitonin gene-related peptide (CGRP) (2). In the absence of CGRP signaling, ILC2 responses and worm expulsion were enhanced. We interpret these data to show that CGRP shapes innate lymphocyte responses to interleukins, alarmins and other neuropeptides during type 2 innate immune responses. JAKs propagate cytokine signals by activating members of a family of transcription factors called STATs (signal transducers and activators of transcription). One important action of cytokines in which STATs play a key role is the differentiation and activation of different subsets of lymphocytes to attain distinct fates, as well as the induction of other cytokines. Our previous work has documented critical functions of STAT5 in CD4+ helper T cells, ranging from suppression of follicular helper T cell function to promotion of regulatory T cells. Using an influenza infection model, this year we report that that while germinal center follicular helper T cells secrete large amounts of IL-2, they respond poorly to this cytokine. We found that this feature was mediated by IL-6, which inhibited induction of IL-2Rb (CD122) by antagonizing STAT5 binding to the Il2rb locus (3). Our previous work has shown that STAT5 has an important role in regulating the CD4 T helper (Th) subset that selectively produces IL-9 (Th9 cells). This year we reported the impact of retinoic acid (RA) on the genome-wide transcriptional response during Th differentiation to multiple subsets, showing that RA effects were subset-selective and were most significant in Th9 cells(4). RA directly targeted the extended Il9 locus, antagonizing the action of STAT5 and broadly modifying the Th9 epigenome through RAR. RA-RAR activity limited murine Th9-associated pulmonary inflammation, and human allergic inflammation was associated with reduced expression of RA target genes. Thus, repression of the Th9 program is a major function of RA-RAR signaling and is likely relevant for IL-9-related diseases, like asthma and allergy. STAT5 is also known to favor the generation and survival of memory T cells. By studying a patient and employing mouse models we found that STAT5 also promotes cell death in memory T cells in mice and humans (5). Extending our studies of T cell memory, we compared single-cell transcriptomes and epigenetic profiles of CD8 T cells responding to acute and chronic viral infections (6). We found that progenitor-like CD8 T cells became distinct from memory precursor cells before the peak of the T cell response. We identified a gene module containing the Tox transcription factor associated with progenitor-like cells. Expression of Tox promoted the persistence of antiviral CD8 T cells and was required for the programming of progenitor-like CD8 T cells. In related collaborative work, we showed that skin commensals such as S. epidermidis elicit IL-17- (Tc17) and IFN-g-(Tc1) producing CD8 T cells (7). Analysis of the transcriptomes of these cells revealed evidence of tissue repair function beyond typical effector functions. This work shows that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota through the specification of CD8 T cells that promote wound healing. We also found that skin-resident commensal-specific T cells harbor a paradoxical program characterized by a type 17 program associated with a poised type 2 state (8). Following injury or exposure to inflammatory mediators such as interleukin-18, these cells rapidly release type 2 cytokines, which promotes tissue repair. These data indicate that commensal-specific T cells co-opt tissue residency and cell-intrinsic flexibility as a means to promote both local immunity and tissue adaptation to injury. ILC3 are an important but heterogeneous lymphoid population in the gut, involved in regulation of barrier homeostasis and inflammation. In this years work, we examined that initial events that can drive ILC3 cells expressing natural cytotoxicity receptors (NCR+ ILC3) to acquire type 1 features (9). We found that NCR+ ILC3 exhibited high basal expression of the STAT4 due to expression of T-BET. As a result, IL-23, by accessing STAT4, induced expression of IL-22, followed by a production of IFN-g, thus altering the balance between a type 3 and type 1 response. Based on our success in using genomic approaches to gain insight in lymphocytes, we also contributed to work with collaborators on myeloid cells. In one project we investigated the roles of classical monocytes and non-classical monocytes in arthritis (10). In a mouse arthritis model, we found that non-classical monocytes are pivotal cells in arthritis tissue damage, displaying increased capacity to differentiate into osteoclasts. Thus, non-classical monocytes might be possible therapeutic targets in prevention of inflammatory joint damage.Myeloid cells also contribute to cardiac repair during myocardial infarction. We identified a specialized macrophage population with a distinct transcriptomic program, loss of which enhanced interstitial fibrosis after ischemic injury (11). These findings reveal an immune cardioprotective role for the pericardial tissue compartment and suggest that removal of the pericardium may need to be reconsidered. In another collaborative study, we examined the role of autophagy in macrophages (12). We identified a macrophage-specific isoform of the vacuolar ATPase protein ATP6V0D2 that promotes autophagosome-lysosome fusion. We generated Atp6v0d2 mice and showed that deficiency of the gene resulted in mitochondrial damage in macrophages, enhanced inflammasome activation and reduced clearance of Salmonella typhimurium.